Carbohydrate molecules known as saccharides or sugars are clinically and physiologically important analytes that are implicated in numerous medical conditions and disorders. Monosaccharides, such as glucose and fructose, are saccharide monomers that form the basic structural units of more complex sugars. Monosaccharides are also clinically significant in their own right, due in part to their role in disorders such as diabetes. Polysaccharides are naturally ubiquitous molecules that are involved in diverse biological systems ranging from plant structure to blood-type grouping. Because of the widespread importance of saccharides, methods for reliably detecting their presence in a broad array of biological, chemical and clinical samples remains an ever-pressing need.
Cell-surface polysaccharides are but one important group of saccharides. As part of glycosylated proteins and lipids, these polysaccharides often form characteristic signatures of different cell types. See, Fukuda, M. (1992) Cell Surface Carbohydrates and Cell Development (Boca Raton: CRC); Fukuda, M. (1994) Cell Surface Carbohydrates: Cell-type Specific Expression. In Molecular Glycobiology, M. Fukuda, & O. Hindsgaul. eds. (New York: Oxford University, pp 1-52).
Certain cell surface carbohydrates, such as sialyl Lewis X (sLex), sialyl Lewis A (sLea), Lewis X (Lex) and Lewis Y (Ley) (structures illustrated in FIG. 1), have been associated with the development and progression of many types of cancers. See, Fukuda, M. (1992) Cell Surface Carbohydrates in Hematopoietic Cell Differentiation and Malignancy. In Cell Surface Carbohydrates and Cell Development, M. Fukuda. ed. (Boca Raton: CRC). pp 127-160; Dennis, J. W. (1992) Changes in Glycosylation Associated with Malignant Transformation and Tumor progression. In Cell Surface Carbohydrates and Cell Development, M. Fukuda. ed. (Boca Raton: CRC). pp 161-194; Jorgensen, T., et al., (1995) Cancer Res. 55, 1817-1819; Idikio, H. A. (1997) Glycoconjugate J. 14, 875-877; and El-Serag, H. B. and Mason, A. C. (1999) New Engl. J. Med. 340, 745-750. The cell-surface expression of these carbohydrates, which are important components of ligands involved in selectin-mediated cell adhesion and inflammatory responses, are specifically associated with the development and progression of human carcinomas such as hepatocellular carcinoma (HCC). See, Shacter, E. and Weitzman, S. A. (2002) Oncology 16, 217-223; Yago, K., et al. (1993) Cancer Res. 53, 5559-5565.
For example, normally differentiated hepatocytes do not express sLex, but chronically diseased liver expresses high levels of sLex, which is associated with a high degree of carcinogenicity. Fujiwara, Y. et al. (2002) Hepatogastroenterology 49, 213-217. Over-expression of sLex in chronic inflammatory diseases of the liver has been reported in several contexts by multiple investigators. Minta, J. O. et al. (1998) Biochim. Biophys. Acta. 1442, 286-295; Okada, Y. et al. (1994) Cancer 73, 1811-1816; and Jezequel-Cuer, M. et al. (1992) Liver 12, 140-146. Loss and gain of sLex expression in variously differentiated HCC specimens has also been well described.
However, the specific role(s) for sLex in transformation and progression to HCC are not entirely understood. Sensor compounds that could sensitively trace this development in vivo would likely further the understanding of hepatocarcinogenesis, in addition to providing new diagnostic and therapeutic approaches. Moreover, diagnosis and staging of HCC is often limited due to inability to detect advanced disease. Treatment of HCC is also impaired by lack of sensitive detection and further by drug-resistance. Nakakura, E. K. and Choti, M. A. (2000) Oncology 14, 1085-1098. Sensor compounds that selectively bind sLex could both recognize occult metastasis and provide targeted delivery of treatment, and thus may improve chances for success in treatment of this disease.
Antibodies specific for cell-surface polysaccharides have been used for the development of in vitro diagnostic and detection tools, targeted drug delivery vectors, and tissue-specific imaging agents. However, success in the in vivo application of antibody-based diagnostic and therapeutic agents has been limited partly because of their poor stability, immunogenicity, poor permeability, and complexity in chemical conjugation with the diagnostic or imaging agents. The development of small, organic molecule-based compounds capable of specific recognition of cell-surface biomarkers would be advantageous, as they generally possess more desirable pharmaceutical, biopharmaceutical, and chemical properties. Such sensor compounds would be useful for diagnostic labeling, drug delivery, and selective imaging applications, and could also be considered antibody mimics for the high specificity recognition of cell biomarkers such as sLex and other cell surface carbohydrates. Unfortunately, the development of selective sensor compounds for polysaccharides such as sLex has been very limited. See, e.g., Sugasaki, A. et al. (2001) J. Am. Chem. Soc. 123, 10239-10244. Possible reasons for this limited development include the complexity of polysaccharides and their conformational flexibility, which makes sensor construction difficult.
Among the monosaccharides, of particular medical and clinical interest is the monosaccharide glucose. The production and the consumption of glucose are regulated such that the concentration of glucose is relatively constant in the body fluids of normal or healthy mammals. A disruption of this regulation of glucose can be associated with diseases such as diabetes and hypoglycemia. One of the major challenges in the treatment of these diseases is the necessity to frequently monitor tissue glucose concentrations. The most commonly used technology for blood glucose concentration determination is an enzyme-based method, which requires frequent collection of blood samples. This is commonly accomplished by drawing a small blood sample (as by a fingerstick) several times daily. A patient typically uses a lancet or needle to draw a droplet of blood and applies the droplet to a reagent strip that is read in a small meter. This approach to glucose monitoring presents several problems, including inconvenience and resulting non-compliance by patients, and the fact that this method is not a continuous monitoring method.
Less invasive methods for measuring glucose in vivo have been described. These methods are generally based on the use of implanted sensor particles capable of generating a detectable analyte signal in response to the analyte concentration in the body. Moreover, there is presently a great deal of interest in the development of continuous glucose monitoring systems, which would be able to provide patients with instantaneous feedback and should help to improve the management of proper glucose concentration in diabetic patients. See, e.g., Koschinsky, T. et al. (2001) Diabetes-Met. Res. Rev. 17, 113-123; Gerritsen, M. et al. (1999) Netherland J. Med., 54, 167-179; Daniloff, G. Y. (1999) Diabetes Tech. Therap. 1, 261-266; Atanasov, P. et al. (1997) Biosen. Bioelectron. 12, 669-680 and Kerner, W. Exp. (2001) Clin. Endocrin. Diab. 109, S341-S346 Suppl. 342. Devices capable of continuous glucose monitoring can be coupled to an insulin delivery device to achieve feedback-controlled delivery of insulin.
To develop a continuous monitoring system, it would be advantageous to use an implantable device that is in constant contact with biological fluid to give a continuous reading of glucose concentration. It is unlikely that the currently used enzyme-based methods could be incorporated into implantable devices, due to instability issues associated with protein-based products. See Gerritsen et al., supra. Non-enzymatic sensor compounds offer the advantage of higher stability and comparatively easy manufacturing. To develop chemical sensor-based continuous monitoring devices, sensor compounds that show high selectivity and appropriate affinity to glucose must be developed.
In view of the foregoing, there remains a need for compounds and methods for selectively detecting a variety of monosaccharides and polysaccharides. The high degree of structural similarity between different saccharides can hinder their selective detection. Color assays for saccharides are known, including those based on certain synthetic molecules and others based on enzymes that are known to bind of cleave saccharides. Enzymatic assays offer generally greater specificity than non-enzymatic color assays, but are usually more expensive and require greater care of reagents. For example, enzymes must be protected from extreme conditions during manufacture, storage and use. Ideally, the detection of saccharides involves compounds that are highly specific, highly selective and employ stable, non-enzymatic reagents.
Critical to the development of high affinity and high specificity sensors for saccharides is the need for recognition moieties that have strong interactions with the functional groups (e.g., hydroxyl groups) of a saccharide. Useful sensor molecules will generally also have a reporter moiety (e.g., a fluorophore), as well as a three-dimensional scaffolding moiety or “switch” that is mediated by a substrate recognition event (e.g., binding of the recognition moiety to a saccharide) and which triggers a reporting event.
With regard to the selection of a recognition moiety for a saccharide sensor compound, boronic acid has been known to have high affinity for diol-containing compounds such as carbohydrates. See Lorand, J. P. and Edwards, J. O. (1959) J. Org. Chem. 24, 769; Sugihara, J. M. and Bowman, C. M. (1958) J. Am. Chem. Soc. 80, 2443; Springsteen, G. and Wang, B. (2002) Tetrahedron 58, 5291-5300. By taking advantage of this strong interaction, several molecular recognition systems for carbohydrates based on boronic acid moieties have been developed. See, e.g., Wang, W. et al. (2002) Current Org. Chem. 6, 1285-1317; Yang, W. et al. (2003) Med. Res. Rev. 23, 346-368; James, T. D. et al. (1995) J. Am. Chem. Soc. 117, 8982-8987; James, T. D. et al. (1995) Nature (London) 374, 345-347; Eggert, H. et al. (1999) J. Org. Chem. 64, 3846-3852; Adhikiri, D. P. and Heagy, M. D. (1999) Tetrahedron Lett. 40, 7893-7896; Wiskur, S. L. and Anslyn, E. V. (2001) J. Am. Chem. Soc. 123, 10109-10110; Tong, A.-J. et al. (2001) Anal. Chem. 73, 1530-1536; Yang, W. et al. (2001) Angew. Chem. Int. Ed. 40, 1714-1718; DiCesare, N. and Lakowicz, J. R. (2001) Org. Lett. 3, 3891-3893; and Ward, C. J. et al. (2002) Org. Lett. 4, 477-479.
As stated above, once the recognition moiety of a sensor compound has bound its saccharide target, the binding should trigger a reporting event. It is known that anthracene fluorescence can be quenched by nitrogen lone pair electrons on an amino group (see schematic in FIG. 2). However, this quenching can be removed or reduced if lone pair electrons are masked through B—N bond formation. See James, T. D. et al. (1995) J. Am. Chem. Soc. 117, 8982-8987; Wulff, G. (1982) Pure Appl. Chem. 54, 2093-2102. Since binding with a carbohydrate is known to increase the acidity of boronic acid, the boronic ester formation will also increase the B—N bond strength, which results in the masking of the nitrogen lone pair electrons. Consequently, the fluorescence intensity of the anthracene system increases (FIG. 2).
Stated another way, the fluorescent intensity of the sensor compound changes in response to photo-induced electron transfer (PET) between the amine group and the fluorophore, as modulated by binding of saccharide hydroxyls to a pair of boronic acids. In the absence of saccharide binding, the fluorescence generated by the fluorescent group is quenched by the unshared electron pair of the nitrogen atom. When saccharide is bound, the unshared electron pair is utilized in the bond formation and does not participate in fluorescence-quenching. Consequently, intrinsic fluorescence of the sensor compound is expressed.
Boronic acid compounds have been used for the synthesis of glucose sensors. Among the significant development in the field are the diboronic acid sensors by the Shinkai group (see, James et al. (1995) supra, and James, T. D. et al. Angew. Chem. Int Ed. Engl. 1996, 35, 1910-1922); the Norrild group (see Norrild, supra; Eggert, supra; and Bielecki, M. et al. (1999) J. Chem. Soc., Perkin Trans. 2, 449-455); and the Drueckhammer group (see Yang (2001), supra). See also U.S. Pat. No. 5,503,770 to James et al.; U.S. Pat. No. 6,387,672 to James et al., U.S. Pat. No. 6,387,672 to Arimori et al., and International Patent Application Publication No. WO 01/20334 to Satcher et al, the disclosures of which are incorporated by reference.
The Shinkai and Norrild sensors showed enhanced fluorescence after binding with sugar, with a modest selectivity for glucose over other carbohydrates. For example, one sensor developed by the Shinkai group exhibited a 12- and 25-fold selectivity for glucose over fructose and galactose, respectively. The Drueckhammer system exhibited much higher selectivity for glucose over fructose and galactose than that of the Shinkai system. However, the fluorescence intensity of the sensor was reduced upon binding with sugars, which may limit its potential application. Recently, the Heagy group reported a monoboronic acid compound that showed the greatest spectroscopic changes with glucose compared to other sugars such as fructose. H Cao et al. (2002) Org. Lett. 4, 1503-1505. However, the binding constant of this compound with glucose was reported to be lower than that of fructose.